Inclusion of restriction sites ...ERRATUM

Michael Coady coady at ERE.UMontreal.CA
Tue Nov 5 12:38:02 EST 1996


aiyar at ebv.oncology.wisc.edu (Ashok Aiyar) writes:

>On Sun, 03 Nov 1996 13:25:29 +0000, Andrei Popov <andrei.popov at bbsrc.ac.uk> wrote:
>>I cloned many a PCR fragment using this approach, and recently also
>>5'RACE products where the percentage of correct clones was exceptionally 
>>high.

>[deleted]

>>
>>I find the tone of Dr. Aiyar's comments simply unprofessional and rude.

>Unprofessional or otherwise, I have tried this cloning approach
>a few years ago and failed on every attempt.  Therefore I did the
>following experiment to test what types of ends Klenow left in
>my hands.


	I've used this technique with great success, but not using
Klenow.  A similar paper was in NAR a few years ago that described
using Exo III to nibble off the ends on the 3' strand.  You've
got to do the exonuclease reaction on ice for about 1 minute, as I
recall.  If people want, I could look up the reference.  Anyways,
this is how we do our PCR now to get handy ends.

Mike


-- 
Michael J. Coady, Ph.D.
COADY at ERE.UMONTREAL.CA
The opinions expressed above are solely those of the author and do not,
in any way, shape or form, represent the Universite de Montreal.



More information about the Methods mailing list